CREBZF Human

What are the key structural features of human CREBZF?

Human CREBZF is a member of the mammalian ATF/CREB family of transcription factors characterized by a basic leucine zipper (bZIP) domain. The gene can produce at least four distinct isoforms through a combination of alternative splicing and alternative translation initiation . The most well-studied isoforms include:

• Short form CREBZF (sZF)

• Long form CREBZF (lZF) with an additional 82 amino acids at the N-terminus

• IFFFR-tailed variants (sZF-IF3R and lZF-IF3R) that contain a unique C-terminal pentapeptide IFFFR tail

Structurally, CREBZF contains specific domains for DNA binding, protein-protein interactions, and post-translational modifications. One particularly important site is Lys208, which undergoes acetylation that regulates protein stability .

How does CREBZF regulate gene expression?

CREBZF functions as a transcription factor through several mechanisms:

• Direct DNA binding: CREBZF can bind to specific DNA elements, including the C/EBP-ATF element found in promoters of target genes like CHOP .

• Protein-protein interactions: CREBZF associates with transcriptional coregulators such as PGC-1α to repress thermogenic gene expression . It also interacts with NFκB signaling components, notably competing with IκBα for binding to p65, which enhances proinflammatory gene transcription .

• Context-dependent functions: Different isoforms of CREBZF appear to have distinct functions, with the sZF-IF3R isoform being particularly potent at inducing CHOP expression and promoting apoptosis during ER stress .

• Post-translational regulation: The transcriptional activity of CREBZF is modulated by acetylation, which affects its protein stability and function .

What experimental techniques are most reliable for detecting CREBZF expression in human tissues?

For reliable detection of CREBZF in human tissues, researchers should consider:

• Isoform-specific quantitative RT-PCR: Given the multiple isoforms produced through alternative splicing, primer design must be specific to distinguish between transcript variants. This is particularly important as different isoforms may have distinct functions .

• Western blotting with isoform-specific antibodies: When performing immunoblotting, researchers should be aware that:

  • Different isoforms have distinct molecular weights

  • The sZF-IF3R isoform is highly unstable with a half-life of approximately 6 hours under normal conditions, reducing to 1.5-2.5 hours under ER stress conditions

  • Antibodies targeting the N-terminus or C-terminus may detect different subsets of isoforms

• Immunohistochemistry/Immunofluorescence: These techniques can provide insights into subcellular localization, which varies among isoforms, with some predominantly nuclear and others showing both nuclear and cytoplasmic distribution .

• Protein stability assays: Cycloheximide chase assays are particularly valuable for studying CREBZF due to its regulated protein stability .

How does CREBZF contribute to insulin resistance mechanisms?

CREBZF has emerged as a significant regulator of insulin resistance through its role in adipose tissue inflammation. Key mechanisms include:

• Macrophage-specific effects: Myeloid-specific knockout of CREBZF in mice fed high-fat, high-sucrose diets leads to attenuated inflammation, improved insulin sensitivity, and better glycemic control, indicating CREBZF's central role in promoting inflammatory responses in macrophages .

• NFκB signaling enhancement: Mechanistically, CREBZF in macrophages enhances NFκB signaling by competitively inhibiting the binding of IκBα to p65. This competition promotes p65 nuclear translocation and increases transcription of proinflammatory genes, creating a chronic inflammatory state that contributes to insulin resistance .

• Response to proinflammatory signals: CREBZF protein levels are significantly induced in macrophages in response to proinflammatory signals, suggesting it functions as part of an amplification loop in chronic inflammation .

• Therapeutic target potential: Bromocriptine has been identified as a small molecule inhibitor of CREBZF in macrophages and is sufficient to suppress diet-induced proinflammatory phenotypes and improve metabolic dysfunction .

What is the role of CREBZF in adipose tissue browning and thermogenesis?

CREBZF serves as an important negative regulator of adipose tissue browning and thermogenesis:

• Glucose-responsive regulation: Glucose induces CREBZF expression in human white adipose tissue (WAT) and inguinal WAT (iWAT) in mice, suggesting CREBZF mediates glucose effects on thermogenic capacity .

• Post-translational regulation: Lys208 acetylation, modulated by CREB-binding protein (CBP)/p300 (as transacetylase) and HDAC3 (as deacetylase), regulates CREBZF protein stability. Glucose-induced acetylation reduces proteasomal degradation and increases CREBZF stability .

• Thermogenic gene repression: CREBZF associates with PGC-1α to repress thermogenic gene expression. Adipose-specific CREBZF knockout (CREBZF FKO) mice display enhanced thermogenic gene expression, increased browning of iWAT, and improved adaptive thermogenesis during cold exposure .

• Inverse correlation with thermogenic markers: Expression levels of CREBZF are negatively correlated with UCP1 in human adipose tissues and are increased in WAT of obese ob/ob mice .

• Energy expenditure modulation: CREBZF-mediated inhibition of energy dissipation likely represents a homeostatic mechanism for regulating energy balance and preventing hyperactivation of thermogenesis .

How should researchers design experiments to investigate CREBZF's tissue-specific functions in metabolism?

When investigating CREBZF's tissue-specific functions in metabolism, researchers should consider:

• Tissue-specific knockout models: Generate tissue-specific knockout mice (e.g., adipose-specific or macrophage-specific) rather than global knockouts to distinguish direct from secondary effects. The myeloid-specific and adipose-specific CREBZF knockout models have already revealed distinct phenotypes .

• Physiological challenges: Expose models to relevant physiological challenges:

  • High-fat, high-sucrose diet for insulin resistance studies

  • Cold exposure for thermogenesis studies

  • Glucose tolerance and insulin sensitivity tests

  • Metabolic cage studies to assess energy expenditure

• Ex vivo and in vitro validation: Complement in vivo studies with:

  • Primary cell isolation (adipocytes, macrophages) from knockout and control mice

  • Co-culture systems to study cell-cell interactions (e.g., macrophages with adipocytes)

  • Human tissue samples to validate findings across species

• Isoform-specific analyses: Given the multiple isoforms of CREBZF, design experiments that can distinguish their potentially distinct functions, using isoform-specific overexpression or knockdown approaches .

• Molecular interaction studies: Investigate protein-protein interactions relevant to each tissue context (e.g., PGC-1α in adipose tissue, NFκB components in macrophages) .

What is the evidence linking CREBZF to cancer progression?

Several lines of evidence connect CREBZF to cancer, particularly breast cancer:

• Tumor suppressor activity: In breast cancer, CREBZF appears to function downstream of the long non-coding RNA MBNL1-AS1 and is negatively regulated by miR-423-5p. CREBZF knockdown impairs the inhibition of cancer cell growth mediated by low miR-423-5p expression, suggesting a tumor suppressor role .

• Signaling pathway involvement: CREBZF influences the PI3K/AKT pathway, which is associated with cell proliferation and apoptosis in breast cancer. The MBNL1-AS1/miR-423-5p/CREBZF axis represents a potential regulatory mechanism in breast cancer development .

• Biomarker potential: The complete understanding of the MBNL1-AS1/miR-423-5p/CREBZF axis in breast cancer suggests it could be used as a biomarker for predicting survival among breast cancer patients .

• Cancer cell phenotype regulation: CREBZF has been shown to inhibit cancer cell proliferation, migration, and invasion in breast cancer models .

How does CREBZF function in the unfolded protein response (UPR) and ER stress?

CREBZF plays important roles in the unfolded protein response (UPR) and ER stress pathways:

• Stress-induced expression: In ER stressor-treated HeLa cells, both CREBZF transcription and the protein level of the lZF-IF3R isoform are continuously induced, peaking at 24-36 hours after stress induction .

• Isoform-specific functions: Only the IFFFR-tailed isoforms (particularly sZF-IF3R) induce CHOP, a key mediator of ER stress-induced apoptosis. The sZF-IF3R isoform induces CHOP through binding to the C/EBP-ATF element in its promoter .

• Protein stability regulation: Under ER stress conditions, the sZF-IF3R isoform becomes highly unstable, with its half-life decreasing from 6 hours in untreated cells to 1.5-2.5 hours in cells treated with ER stressors like tunicamycin or thapsigargin .

• Apoptotic signaling: Overexpression of the sZF-IF3R isoform promotes apoptosis, implicating it in the CHOP-mediated apoptotic signaling pathway. This suggests a role for CREBZF in determining cell fate during prolonged ER stress .

• Temporal regulation: The late onset expression of sZF-IF3R appears essential for its function in the mammalian UPR, suggesting it acts in the later phases of the stress response when cells transition from adaptation to apoptosis .

What methodological approaches are most effective for studying CREBZF in disease models?

For studying CREBZF in disease models, researchers should consider:

• Disease-relevant cellular systems:

  • Immortalized cell lines with appropriate tissue origins

  • Primary cells isolated from patients or disease models

  • 3D organoid cultures that better recapitulate tissue architecture

• Genetic manipulation strategies:

  • CRISPR/Cas9-mediated gene editing for complete knockout or specific mutations

  • shRNA or siRNA for transient knockdown studies

  • Isoform-specific targeting strategies given the multiple CREBZF variants

  • Rescue experiments with wild-type or mutant constructs to establish causality

• Conditional and inducible systems:

  • Tet-on/off systems for controlled expression

  • Cre-loxP systems for tissue-specific and temporal control in mouse models

  • Stress induction protocols (e.g., tunicamycin or thapsigargin for ER stress)

• Multiomics approaches:

  • RNA-seq to identify transcriptional targets

  • ChIP-seq to map genome-wide binding sites

  • Proteomics to identify interaction partners and post-translational modifications

  • Metabolomics to assess metabolic consequences

• Translational approaches:

  • Patient sample analysis for correlation with disease progression

  • Preclinical testing of interventions targeting CREBZF (e.g., bromocriptine in metabolic disease)

What are the key considerations when designing CREBZF overexpression or knockdown experiments?

When designing CREBZF overexpression or knockdown experiments, researchers should consider:

• Isoform specificity:

  • Target specific isoforms (sZF, lZF, sZF-IF3R, lZF-IF3R) as they may have distinct functions

  • Design constructs that exclusively express one isoform

  • For knockdown, carefully design siRNA/shRNA to target either common regions or isoform-specific sequences

• Expression level control:

  • Use inducible systems to avoid potential toxicity from constitutive overexpression

  • Consider the natural expression levels and avoid supraphysiological overexpression

  • Monitor protein stability, especially for sZF-IF3R which has a short half-life (6 hours normally, 1.5-2.5 hours under ER stress)

• Cellular context:

  • Select appropriate cell types (macrophages for inflammation studies, adipocytes for metabolic studies)

  • Consider the endogenous expression levels in your chosen cell type

  • Account for potential cell type-specific interacting partners

• Functional readouts:

  • Include positive controls to validate successful manipulation

  • Measure known downstream targets (e.g., CHOP expression for sZF-IF3R)

  • Assess relevant cellular phenotypes (e.g., inflammation, thermogenesis, apoptosis)

• Rescue experiments:

  • Perform rescue experiments with wild-type and mutant constructs (e.g., K208R mutation that prevents acetylation)

  • Use isoform-specific rescue to determine functional redundancy

How can researchers accurately assess CREBZF protein-protein interactions?

For accurate assessment of CREBZF protein-protein interactions, researchers should:

• Co-immunoprecipitation approaches:

  • Use antibodies against endogenous proteins when possible

  • Consider epitope tagging (FLAG, HA, etc.) when specific antibodies are unavailable

  • Include appropriate controls (IgG control, input samples)

  • Perform reciprocal co-IPs to confirm interactions

  • Use crosslinking for transient or weak interactions

• Proximity labeling methods:

  • BioID or TurboID fusion proteins to identify proximal proteins in living cells

  • APEX2 proximity labeling for temporal control and subcellular specificity

  • These approaches can capture both stable and transient interactions

• Fluorescence-based interaction assays:

  • Fluorescence resonance energy transfer (FRET)

  • Bimolecular fluorescence complementation (BiFC)

  • These provide spatial information about interactions within cells

• Domain mapping:

  • Generate truncation and point mutants to map interaction domains

  • Focus on known functional domains and post-translational modification sites (e.g., K208)

• In vitro binding assays:

  • GST pulldown or in vitro transcription/translation for direct interaction assessment

  • Surface plasmon resonance (SPR) for binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Physiological relevance validation:

  • Confirm interactions under relevant physiological conditions (e.g., ER stress, cold exposure)

  • Investigate how interactions change with stimuli or in disease states

  • Focus on known interaction partners like PGC-1α and NFκB components

What cellular assays best measure CREBZF's impact on inflammation and metabolism?

To effectively measure CREBZF's impact on inflammation and metabolism, researchers should employ:

• Inflammation assays:

  • Cytokine production measurement (ELISA, multiplex assays)

  • NFκB reporter assays to monitor signaling pathway activation

  • Flow cytometry to assess macrophage polarization states (M1 vs. M2)

  • Macrophage migration and invasion assays

  • Co-culture systems with adipocytes to study tissue-level inflammatory responses

• Insulin sensitivity measurements:

  • Glucose uptake assays in adipocytes or muscle cells

  • Insulin signaling pathway activation (phosphorylation of AKT, IRS1)

  • Lipolysis assays in adipocytes

  • Glucose production assays in hepatocytes

• Thermogenesis and browning assays:

  • Oxygen consumption rate (OCR) measurements

  • Expression analysis of thermogenic genes (UCP1, PGC-1α, PRDM16)

  • Mitochondrial content and activity assays

  • Lipid droplet staining and analysis

  • Seahorse extracellular flux analysis for metabolic profiling

• ER stress and apoptosis assays:

  • UPR pathway activation markers (phospho-PERK, phospho-IRE1α, XBP1 splicing)

  • CHOP expression and localization

  • Caspase activity assays

  • Annexin V/PI staining for apoptosis quantification

  • Calcium flux measurements

• Gene expression readouts:

  • Reporter assays for specific promoters (e.g., CHOP promoter with C/EBP-ATF element)

  • ChIP assays to assess transcription factor binding

  • RNA-seq for genome-wide transcriptional effects

How can researchers reconcile seemingly contradictory findings about CREBZF's role in different tissue contexts?

When addressing seemingly contradictory findings about CREBZF functions across tissues:

• Isoform-specific effects: Different tissues may preferentially express specific CREBZF isoforms with distinct functions. For example, the IFFFR-tailed sZF-IF3R isoform is particularly potent at inducing CHOP and promoting apoptosis, while other isoforms may have different effects . Researchers should:

  • Characterize isoform expression patterns across tissues

  • Perform isoform-specific gain and loss of function studies

  • Identify isoform-specific interacting partners

• Context-dependent protein interactions: CREBZF interacts with different partners depending on cellular context:

  • In adipocytes, CREBZF associates with PGC-1α to repress thermogenic gene expression

  • In macrophages, CREBZF interacts with NFκB signaling components

  • In cells under ER stress, CREBZF regulates CHOP through C/EBP-ATF elements

• Post-translational modification differences: CREBZF undergoes acetylation at K208, which affects its stability and function . This modification may be differentially regulated across tissues, creating context-specific functions.

• Temporal dynamics: CREBZF shows complex temporal regulation, particularly during stress responses. The sZF-IF3R isoform is highly unstable under ER stress with its half-life decreasing significantly . Researchers should:

  • Perform detailed time-course experiments

  • Consider the temporal sequence of cellular responses

  • Account for protein turnover rates in different contexts

• Integrated multi-tissue approaches: To reconcile contradictory findings, researchers should employ:

  • Systems biology approaches to model tissue-specific networks

  • Multi-tissue analyses in the same experimental system

  • Conditional tissue-specific knockout models with appropriate controls

What are the emerging techniques for studying CREBZF post-translational modifications and their functional consequences?

Emerging techniques for studying CREBZF post-translational modifications include:

• Site-specific lysine acetylation analysis:

  • Targeted mass spectrometry approaches for quantitative analysis of K208 acetylation

  • Development of acetylation-specific antibodies

  • CRISPR knock-in of acetylation-mimetic mutations

  • Quantitative proteomics with heavy isotope-labeled peptide standards

• Proximity-dependent labeling for identifying modification enzymes:

  • TurboID or miniTurbo fusions to identify nearby acetyltransferases/deacetylases

  • APEX2-based approaches for temporal control

  • These approaches could help identify additional enzyme regulators beyond the known CBP/p300 and HDAC3

• Live-cell imaging of modification dynamics:

  • FRET-based biosensors for acetylation

  • Fluorescent fusion proteins to track stability and localization

  • Optogenetic control of modification enzymes for precise temporal manipulation

• Single-cell analysis of modification heterogeneity:

  • CyTOF with modification-specific antibodies

  • Single-cell proteomics approaches

  • These reveal cell-to-cell variation in modification states

• Engineered cellular systems:

  • Orthogonal acetylation systems to specifically target CREBZF

  • Degron-based systems to control protein stability in response to small molecules

  • These allow precise manipulation of protein levels and modifications

• Computational approaches:

  • Molecular dynamics simulations to predict structural consequences of acetylation

  • Machine learning to identify patterns in modification data

  • Network analysis to predict functional consequences of modifications

How might CREBZF research inform therapeutic approaches for metabolic diseases?

CREBZF research has significant implications for therapeutic approaches in metabolic diseases:

• Macrophage-targeted anti-inflammatory strategies:

  • CREBZF inhibition in macrophages could reduce adipose tissue inflammation and improve insulin sensitivity

  • Bromocriptine has been identified as a CREBZF inhibitor that suppresses diet-induced inflammation and improves metabolic dysfunction

  • Development of macrophage-specific delivery systems could enhance efficacy while reducing off-target effects

• Thermogenic activation approaches:

  • Inhibiting CREBZF in adipose tissue could enhance browning and thermogenesis, potentially increasing energy expenditure

  • This represents a novel approach to weight management in obesity

  • Combination with existing metabolic therapies might produce synergistic effects

• Personalized medicine considerations:

  • CREBZF expression or activity might vary among individuals with metabolic diseases

  • Genetic or epigenetic variation in the CREBZF gene could predict response to therapies

  • Development of biomarkers based on CREBZF pathway activity

• Potential therapeutic modalities:

  • Small molecule inhibitors targeting CREBZF directly or its interactions with partners

  • Antisense oligonucleotides or siRNAs for targeted knockdown

  • Gene therapy approaches for tissue-specific modulation

• Considerations for clinical translation:

  • Potential for tissue-specific effects and unintended consequences (e.g., apoptosis induction through CHOP)

  • Need for careful assessment of safety given CREBZF's role in multiple pathways

  • Development of appropriate biomarkers to monitor target engagement and efficacy